SUMMARY Most human cancer cells exhibit genome instability, ranging from elevated mutation rates (base substitutions and indels) to chromosomal rearrangements (CRs) and aneuploidy. The application of next generation sequencing (NGS) technologies to analyze cancer genomes has resulted in a wealth of information on the spectrum of genomic instability, and led to the identification of specific mutation and CR signatures associated with loss of different DNA repair pathways. The prevailing view is that CRs are generated through error-prone processing of damaged chromosomes. Although the nature of the initiating lesions for CRs is unknown, much of the genetic evidence from yeast and human cells implicates DNA replication errors as a source of the broken chromosomes that fuel CRs. The two aims outlined in this proposal will address the source of initiating lesions for CRs, and measure the frequency and spectra of CRs in wild type and repair-deficient cells in response to defined DNA damage. In the first aim, we will induce site-specific DSBs using CRISPR-Cas9 in yeast or human cells and determine the full spectrum of CRs in surviving cells. The experiments will be performed in cells lacking specific components of non-homologous end joining (NHEJ), homology-dependent repair (HR) or DNA damage signaling pathways to determine how distinct types of CRs are suppressed. We expect to define unique CR signatures for each deficiency that could guide identification of novel CR signatures in tumor DNA. In the second aim, we will compare the types of CRs formed in response to a stalled replication fork with CRs resulting from endonuclease-induced DSBs. We will use the bacterial Tus/Ter system to create a site-specific stalled replication fork in yeast or human cells, identify the resulting types of CRs and the factors that suppress different CR outcomes. The powerful genetic screens proposed should enable identification of mutational events (base substitutions and indels) in addition to CRs and we will also characterize these events and the repair pathways that suppress their formation. We expect the knowledge garnered from these studies will aid in identifying new mutational signatures and their underlying pathologies.